Method of judging cardiotoxicity of anthracycline-type anticancer chemical therapeutic by detecting human h-fabp and reagent therefor

ABSTRACT

The present invention provides a method of determining toxicity to the heart of an anthracycline-type anticancer chemotherapeutic agent such as adriamycin etc., which comprises detecting human H-FABP (Human Heart-type Fatty Acid-Binding Protein) in the blood separated from human, a reagent therefor, a kit therefor and the like.

TECHNICAL FIELD

This invention relates to a method of determining toxicity to the heartof anthracycline-type anticancer chemotherapeutic agents, of whichparticularly Doxorubicin hydrochloride (in the present specification,“adriamycin” which is a popular name of the present compound is used)(hereinafter sometimes to be referred to as “cardiotoxicity”), a reagentfor the determination and the like.

BACKGROUND ART

The anthracycline-type anticancer chemotherapeutic agent is a glycosideconsisting of an aglycon part comprising a 4-membered ring quinonestructure as a basic skeleton and saccharides mainly consisting of anamino sugar. As a pharmaceutical agent of this type, for example,adriamycin, daunorubicin hydrochloride, epirubicin hydrochloride,idarubicin hydrochloride, pirarubicin hydrochloride, aclarubicinhydrochloride and the like having the following structures can bementioned.Examples of Anthracycline-Type Anticancer Chemotherapeutic Agents

For cancer treatment, prolonged administration of anthracycline-typeanticancer chemotherapeutic agents is generally employed. Whileanthracycline-type anticancer chemotherapeutic agents have a wide rangeof anticancer spectrum, they are known to show cardiotoxicity as acommon side effect due to myocardial injury action.

As a method of determining toxicity to the heart of ananthracycline-type anticancer chemotherapeutic agent, anelectrocardiogram analysis, a blood biochemical test comprisingmeasurement of Creatine Kinase (CK) in blood, an echocardiogram analysisand the like, which are general tests of cardiac function, areconventionally known and performed. However, since electrocardiogramanalysis and echocardiogram analysis do not specifically detectcardiotoxicity of anthracycline-type anticancer chemotherapeutic agents,they do not have sufficient sensitivity to pick up the initial stage ofthe onset of toxicity of the agents, and can detect only the advancedcardiotoxicity. In addition, only a small amount of creatine kinaseflows (escapes) into the blood due to the cardiotoxicity induced byanthracycline-type anticancer chemotherapeutic agents and creatinekinase requires a long time before escape, and therefore, a problem inclinical situation has existed in that cardiotoxicity ofanthracycline-type anticancer chemotherapeutic agents is not preciselyreflected.

On the other hand, as a myocardial injury marker that detects acutemyocardial infarction and the like, Troponin T (TnT), Myosin Light ChainI (MLC-I), human H-FABP (Human Heart-type Fatty Acid-Binding Protein)and the like have been known.

Human H-FABP is abundant in myocardial cytoplasm, capable of bindingwith fatty acid and is considered to be involved in the intracellulartransport of fatty acid. Human H-FABP is reported to be a proteinconsisting of 132 amino acids and has a molecular weight of 14,768(Biochem. J. (1988) 252 191-198).

With regard to Human H-FABP, JP-A-4-31762 discloses that this protein isuseful as a marker of acute myocardial infarction. However, theabove-mentioned publication does not at all refer to the relationshipbetween the cardiotoxicity of anthracycline-type anticancerchemotherapeutic agents and Human H-FABP.

Therefore, the relationship between human H-FABP and the cardiotoxicityof anthracycline-type anticancer chemotherapeutic agent has not beenknown at all to the present day.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a novel method ofdetermining the toxicity to the heart of anthracycline-type anticancerchemotherapeutic agents such as adriamycin and the like and a reagentfor the determination.

To solve these problems, the present inventors have compared and studiedthe levels of Myosin Light Chain I, Troponin T and human H-FABP in theblood of cancer patients who have been administered with adriamycin,which is a representative anthracycline-type anticancer chemotherapeuticagent, and with whom the expression of cardiotoxicity has been confirmedby electrocardiogram analysis and echocardiogram analysis, and foundthat, while all these markers belong to myocardial injury markers todetect acute myocardial infarction and the like, only the level of humanH-FABP has increased to not less than the cut-off value of acutemyocardial infarction unique to each marker, which resulted in thecompletion of the present invention.

Therefore, the present invention provides the following.

[1] A method of determining toxicity to the heart of ananthracycline-type anticancer chemotherapeutic agent, which comprisesdetecting human H-FABP in the blood separated from human.

[2] The method of [1] above, wherein the detection of human H-FABP isperformed by an immunochemical method using an antibody that recognizeshuman H-FABP.

[3] The method of [2] above, wherein the immunochemical method is anenzyme immunochemical method, a latex agglutination assay or animmunochromatographic assay.

[4] The method of [2] above, wherein the antibody is a monoclonalantibody.

[5] The method of [1] above, wherein the anthracycline-type anticancerchemotherapeutic agent is adriamycin or daunorubicin hydrochloride.

[6] A reagent for determining toxicity to the heart of ananthracycline-type anticancer chemotherapeutic agent, which is used forperforming the method of any of [1] to [5] above.

[7] A reagent for determining toxicity to the heart of ananthracycline-type anticancer chemotherapeutic agent, which comprises anantibody that recognizes human H-FABP.

[8] The reagent of [7] above, wherein the antibody is a monoclonalantibody.

[9] The reagent of [7] above, wherein the anthracycline-type anticancerchemotherapeutic agent is adriamycin or daunorubicin hydrochloride.

[10] A commercial package, comprising the reagent of any of [7] to [9]above, and a written matter associated therewith, the written matterstating that said reagent can or should be used for determining toxicityto the heart of an anthracycline-type anticancer chemotherapeutic agent.

[11] A kit for determining toxicity to the heart of ananthracycline-type anticancer chemotherapeutic agent, which comprises anantibody that recognizes human H-FABP.

[12] The kit of [11] above, wherein the antibody is a monoclonalantibody.

[13] The kit of [11] above, wherein the anthracycline-type anticancerchemotherapeutic agent is adriamycin or daunorubicin hydrochloride.

[14] Use of an antibody that recognizes human H-FABP for determiningtoxicity to the heart of an anthracycline-type anticancerchemotherapeutic agent.

[15] The use of [14] above, which comprises detecting human H-FABP inthe blood separated from human.

[16] The use of [15] above, wherein the detection of human H-FABP isperformed by an enzyme immunochemical method, a latex agglutinationassay or an immunochromatographic assay.

[17] The use of [14] above, wherein the antibody is a monoclonalantibody.

[18] The use of [14] above, wherein the anthracycline-type anticancerchemotherapeutic agent is adriamycin or daunorubicin hydrochloride.

The present invention provides a method of determining toxicity to theheart of an anthracycline-type anticancer chemotherapeutic agent, whichcomprises detecting human H-FABP in the blood separated from human.According to this determination method, determination of thecardiotoxicity of the patients under medication with anthracycline-typeanticancer chemotherapeutic agents such as adriamycin and the likebecomes possible. To be specific, by drawing blood from theabove-mentioned patients, comparing the level of Human H-FABP containedtherein with that of human H-FABP contained in the blood of healthyvolunteers, and further by comparing with cut-off value for thedetermination of acute myocardial infarction, whether or not thecardiotoxicity has been expressed, and when it has been expressed, thelevel of the toxicity can be determined.

In addition, the present invention also provides a reagent fordetermining toxicity to the heart of an anthracycline-type anticancerchemotherapeutic agent, which is used for performing the determinationmethod of the present invention, and the like. This reagent for thedetermination and the like are directly used for practicing thedetermination method of the present invention, and achieve the sameobject as the determination method of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention relates to a method of determining toxicity to theheart of an anthracycline-type anticancer chemotherapeutic agent, whichcomprises detecting human H-FABP in the blood separated from human.

The cardiotoxicity to be the target of determination in the presentinvention arises from the side effect of an anthracycline-typeanticancer chemotherapeutic agent. Examples of the above-mentionedchemotherapeutic agent include conventionally known various agents suchas adriamycin, daunorubicin hydrochloride, epirubicin hydrochloride,idarubicin hydrochloride, pirarubicin hydrochloride, aclarubicinhydrochloride and the like. The determination method of the presentinvention is particularly preferable for determining the cardiotoxicitydue to adriamycin or daunorubicin hydrochloride, from among theseanthracycline-type anticancer chemotherapeutic agents.

In the present invention, by the “determination of toxicity” is meantnot only assumption of the presence or absence of expressed toxicity,but also assumption of the level of toxicity when toxicity is present.

While the determination of toxicity may depend exclusively on thedetection of Human H-FABP, other known methods for the detection ofabnormality in the heart, such as electrocardiogram analysis,echocardiogram analysis and the like may be used in combination todetermine toxicity. By combining plural test methods, the toxicity canbe determined more accurately.

A method of detecting human H-FABP in the blood separated from human inthe present invention is not particularly limited, and may be anymethod; for example, conventionally known methods for quantification,semi-quantification or qualitative analysis, such as immunochemicalmethod, fatty acid binding activity measurement method, variouschromatographies (e.g., HPLC and the like), and the like.

Of these, the detection is particularly preferably performed by a methodaiming at quantification of human H-FABP because the level and progressof cardiotoxicity can be determined, and the detection is particularlypreferably performed by an immunochemical method utilizing an antibodyrecognizing human H-FABP (hereinafter sometimes to be referred to as an“anti-human H-FABP antibody”).

When human H-FABP is detected by an immunochemical method, animmunochemical method using any of a monoclonal antibody and apolyclonal antibody as an anti-human H-FABP antibody can be preferablyused. In view of stable supply of antibody and specificity to humanH-FABP, an immunochemical method using a monoclonal antibody ispreferably used. Anti-human H-FABP antibody itself is known, and can beproduced according to the production method described in JP-A-4-31762 ora method analogous thereto.

The immunochemical method is not particularly limited, and, for example,a conventionally known Enzyme Immunoassay (EIA), Latex AgglutinationAssay, Immunochromatographic Assay, Radio Immunoassay (RIA),Fluorescence Immunoassay (FIA), Luminescence Immunoassay, SpinImmunoassay and the like can be mentioned. Of these, EIA, LatexAgglutination Assay and Immunochromatographic Assay are preferable. InEIA, sandwich Enzyme-Linked Immunosorbent Assay (sandwich ELISA) usingtwo kinds of antibodies, particularly monoclonal antibodies, isparticularly preferable in view of specificity to antigen (human H-FABP)and easiness of detection operation.

When the above-mentioned human H-FABP is to be detected by sandwichELISA, for example, according to the description in Journal ofImmunological Methods 178 (1995) 99-111 and the like, human H-FABP isinserted (sandwiched) between two kinds of antibodies that recognizedifferent epitopes of human H-FABP, i.e., solid phase antibody andenzyme-labeled antibody, and the enzyme level of the labeled antibodythat was bound with human H-FABP is determined, whereby quantitativedetection is performed.

The Latex Agglutination Assay is an immunochemical method utilizing anagglutination reaction between an antibody sensitized Latex particle andan antigen. When the above-mentioned human H-FABP is to be detected bythe Latex Agglutination Assay, quantitative detection is performed bymeasuring the level of agglutination.

In the Immunochromatographic Assay, all immunochemical reaction systemsare retained on a sheet carrier, wherein the operation is completed onlyby the addition (dropwise addition) of blood. In other words, when theblood is added to the carrier, human H-FABP in the blood is bonded to ananti-human H-FABP antibody labeled with gold colloid and the like, andthis bonded form is chromatographically developed on the carrier andcaptured at a particular site (determination region) by a differentsolid phased anti-human H-FABP antibody. When the above-mentioned humanH-FABP is to be detected by this Immunochromatographic Assay, becausethe bonded form is captured as mentioned above, the accumulated samplematerials can be detected by visual observation. Since this method doesnot require a special measuring instrument for practicing, it is anadvantageous method when a rapid treatment becomes necessary dependingon the determination results.

As measurement methods besides the above-mentioned that utilizesspecificity of an anti H-FABP antibody, nephelometry comprisingmeasurement of turbidity associated with an antigen-antibody complexformation, an enzyme sensor electrode method comprising detection ofelectrical potential change due to antigen bond, which utilizes anantibody solid phase membrane electrode, immunoelectrophoresis, westernblotting and the like can be mentioned. Any of these methods sufficesfor detection of human H-FABP.

As the blood for the detection of human H-FABP in the present invention,any blood can be used as long as it is isolated from human, and may beany of whole blood, blood serum and blood plasma. The above-mentionedblood can be obtained appropriately by a treatment according to aconventional method per se.

The determination of toxicity to the heart of an anthracycline-typeanticancer chemotherapeutic agent such as adriamycin and the like can beperformed by comparing the human H-FABP level in the blood detected inthis manner with the human H-FABP level in the blood of healthyvolunteers, and by comparing with a cut-off value of myocardiopathy(e.g., acute myocardial infarction).

For example, according to Okamoto et al., Clinical Chemistry andLaboratory Medicine 2000 38(3) 231-238, average value of the humanH-FABP level in the blood (blood serum) of healthy volunteer is 2.8ng/mL (upper limit being 5.3 ng/mL), and the cut-off value of acutemyocardial infarction, or the level that showed the highest diagnosticefficiency (value satisfying high true positive rate (diagnosticsensitivity) and high true negative rate (diagnostic specificity)) isset to 6.2 ng/mL.

Therefore, when human H-FABP level in the blood of a cancer patientunder medication with an anthracycline-type anticancer chemotherapeuticagent exceeds the upper limit of human H-FABP level in the blood ofhealthy volunteer, 5.3 ng/mL, the presence of suspect of expression ofcardiotoxicity can be determined, and when it increases to the cut-offvalue of acute myocardial infarction, i.e., not less than 6.2 ng/mL, thepossibility of expression of cardiotoxicity can be determined to beextremely high.

The determination results thus obtained are useful for a physician todecide, for a cancer patient under medication with an anthracycline-typeanticancer chemotherapeutic agent, if (1) the administration of the sameagent is to be continued, (2) the administration is to be stopped (thekind (type) of anticancer agent is changed) or (3) the dose is to beincreased or decreased, and the like, and for a patient for whomadministration of an anthracycline-type anticancer chemotherapeuticagent was once stopped due to the expression of cardiotoxicity, it isuseful for determining if (4) administration of this agent is to beresumed.

To be specific, during the administration period of adriamycin, forexample, blood is taken from a patient at a frequency of at least once amonth and human H-FABP level is measured using the obtained blood as asample. In this case, when a human H-FABP level shows not less than 6.2ng/mL, which is a cut-off value of acute myocardial infarction,administration of adriamycin is quickly stopped, and an anticancer agentof a different kind (type) is employed. Since human H-FABP level isconsidered to also reflect the level of cardiotoxicity due toadriamycin, when human H-FABP exceeds 6.2 ng/mL and the value is at ahigh level, the injury the cardiac muscle suffered from is considered tobe large. Thus, it is determined that the administration of the agent isto be stopped and a quick protective measure of cardiac muscle needs tobe taken.

In contrast, when human H-FABP level is less than 6.2 ng/mL,particularly less than 5.3 ng/mL, it is determined that administrationof adriamycin can be continued without a problem.

The present invention also relates to a reagent for determining toxicityto the heart of an anthracycline-type anticancer chemotherapeutic agent,which is used to practice the above-mentioned determination method.Thus, the reagent for determination of the present invention is directlyused for the practice of the aforementioned determination method of thepresent invention, and achieves the same object as the determinationmethod of toxicity.

In addition, the present invention provides a reagent and a kit fordetermining toxicity to the heart of an anthracycline-type anticancerchemotherapeutic agent at least containing an antibody recognizing HumanH-FABP (anti-human H-FABP antibody). Such reagent and a kit fordetermination can be preferably used for, among the aforementioneddetermination methods of the present invention, detection of humanH-FABP by an immunochemical method in blood separated from human.

The anti-human H-FABP antibody can be produced according to a knownmethod, such as the method described in, for example, the aforementionedJP-A-4-31762, and can be applied to an immunochemical method in a freestate, a labeled state or a solidified state. The anti-human H-FABPantibody may be a polyclonal antibody or a monoclonal antibody, but amonoclonal antibody is preferable because specificity and uniformity ofantibody are high. A polyclonal antibody can be produced by immunizingan animal such as mouse, rat, rabbit and the like with human H-FABPtogether with a suitable adjuvant, taking blood and processing the bloodby known treatments. In addition, a monoclonal antibody can be producedby harvesting spleen cells of an animal immunized in this way,subjecting the cells to cell fusion with myeloma cells, and toantibody-producing cells screening and cloning etc. by the method ofMilstein et al., thereby establishing an anti-human H-FABPantibody-producing cell line, and culturing the cell line. Here, humanH-FABP to be used as an immunizing antigen is not necessarily a humancardiac muscle organ derived, natural H-FABP, and may be a recombinanthuman H-FABP obtained by genetic engineering technique or any materialhaving the same effect (fragment).

When the thus-obtained anti-human H-FABP antibody is applied to sandwichELISA, this antibody is produced in the form of a solid phasedanti-human H-FABP antibody or an enzyme-labeled anti-human H-FABPantibody.

A solid phased anti-human H-FABP antibody can be produced by binding anantibody obtained as mentioned above to a solid phase (e.g., microplatewell or plastic beads). Binding to a solid phase can be generallyperformed by dissolving an antibody in a suitable buffer solution suchas citrate buffer solution and the like, and bringing a solid phasesurface in contact with an antibody solution for a suitable time (1-2days). Then, phosphate buffer containing Bovine Serum Albumin (BSA),bovine milk protein and the like is brought into contact with the solidphase to coat a part on the surface of the solid phase that was notcoated with the antibody with the aforementioned BSA and the like.

An enzyme-labeled anti-human H-FABP antibody can be produced by binding(labeling) an anti-human H-FABP antibody, which recognizes an epitopedifferent from the above-mentioned solid phased antibody, with anenzyme. As the enzyme to label the antibody, alkaline phosphatase,glucose oxidase, peroxidase, β-galactosidase and the like can bementioned. The binding between these enzymes and anti-human H-FABPantibody can be performed by a method known per se, such asglutaraldehyde method, maleimide method and the like. In addition, amethod using an avidin-biotin reaction (method comprising reacting anantibody labeled with biotin instead of an enzyme with human H-FABP inblood and then binding with an enzyme-labeled streptavidin) may beemployed.

In sandwich ELISA, enzyme substrate, washing solution, reaction-stoppingsolution, substrate dissolution solution, standard antigen (humanH-FABP) and the like are used as necessary, besides anti-human H-FABPantibody. The present invention may be realized in the form of a kit(kit for determining toxicity to the heart of an anthracycline-typeanticancer chemotherapeutic agent) containing these as components,besides the above-mentioned anti-human H-FABP antibody.

As the enzyme substrate, one suitable for a selected labeled enzyme isused. For example, when alkaline phosphatase is selected as an enzyme,p-nitrophenylphosphate (PNPP) and the like are mentioned, and as a colordeveloping agent in this case, o-phenylenediamine (OPD),tetramethylbenzidine (TMB) and the like are used. As the washingsolution, reaction-stopping solution and substrate dissolution solution,conventionally known ones are appropriately used without any particularlimitation, depending on the selected labeled enzyme.

A specific production method of the reagent for determination of thepresent invention based on sandwich ELISA is described in Ohkaru et al.,Journal of Immunological Methods 178 (1995) 99-111. In addition, a bloodserum human H-FABP measurement kit (“MARKIT (registered trademark) MH-FABP”) s based on sandwich ELISA as a measurement principle has beenavailable from DAINIPPON PHARMACEUTICAL CO., LTD., and this kit can beused as a determination kit relating to the present invention.

In addition, a reagent that detects human H-FABP by Latex AgglutinationAssay is also known and its production method is also specificallydescribed in, for example, Markus Robers et al., Clinical Chemistry 44,No. 7, (1998) 1564-1567 and the above-mentioned JP-A-4-31762.

It is also possible to apply an Immunochromatographic Assay to thereagent for determination of the present invention. Specific productionmethod of the reagent for determination of the present invention byImmunochromatographic Assay is described in Watanabe et al., ClinicalBiochemistry 34 (2001) 257-263. Furthermore, a whole blood human H-FABPdetection reagent (“RAPICHECK (registered trademark) H-FABP”) based onImmunochromatographic Assay as a detection principle has been availablefrom DAINIPPON PHARMACEUTICAL CO., LTD., and this can be used as areagent for determination relating to the present invention.

The present invention is explained in detail by referring to Examplesand Reference Examples, which are not to be construed as limitative.

EXAMPLE 1

The blood from a cancer patient administered with adriamycin was takenthree times during 5 months of adriamycin administration (second bloodsampling was about 2.5 months after the first sampling, third bloodsampling was 20 days after the second sampling) and blood serum wasobtained by a conventional method. Using the obtained blood serum as asample, the level of human H-FABP, Myosin Light Chain I and Troponin Twas measured.

Human H-FABP was measured using a blood serum human H-FABP measurementkit “MARKIT (registered trademark) M H-FABP” (manufactured by DAINIPPONPHARMACEUTICAL CO., LTD.) based on sandwich ELISA using two kinds ofspecific monoclonal antibodies as a measurement principle.

That is, a 1:1 volume mixture (100 μL) of a diluting buffer solution(composition: 0.2% BSA-0.9% NaCl-0.1 mol/L potassium phosphate buffer,pH 7.0) and a blood serum sample were dispensed to a microplate well(antibody bonded well), in which one kind of anti-human H-FABPmonoclonal antibody had been solid phased, and the reaction was carriedout at room temperature (25° C.) for 30 min (first antigen-antibodyreaction). After the reaction, the antibody bonded well was washed 3times with 300 μL of washing solution, then horseradishperoxidase-labeled anti-human H-FABP monoclonal antibody (100 μL) wasdispensed thereto and the reaction was carried out at room temperaturefor 30 min (second antigen-antibody reaction). After washing in the samemanner as above, an enzyme substrate (100 μL, hydrogenperoxide-containing OPD) was added to start the enzyme reaction, whichwas carried out at room temperature for 15 min. A reaction-stoppingsolution (100 μL, 0.9 mol/L sulfuric acid) was added to stop thereaction. As a control, the above-mentioned diluting buffer solution andeach standard solution prepared to make the final concentration of humanH-FABP 0 ng/mL, 5 ng/mL, 10 ng/mL, 25 ng/mL, 50 ng/mL, 100 ng/mL, 250ng/mL were mixed at a volume ratio of 1:1 and processed in the samemanner as above.

The level of color development of each well was determined by measuringan absorbance at 492 nm, and comparing with a standard curve drawn fromthe results of a standard solution containing human H-FABP at eachconcentration employed for the control, whereby the human H-FABP levelsin the blood serum samples were read. In addition, the cut-off value ofacute myocardial infarction for the above-mentioned blood serum humanH-FABP measurement kit was set for 6.2 ng/mL.

For the measurement of Myosin Light Chain I, sandwich RIA was used andfor the measurement of Troponin T, sandwich ELISA was used. The cut-offvalues of acute myocardial infarction in this case were set to 2.5 ng/mLfor Myosin Light Chain I, and 0.1 ng/mL for Troponin T.

The state of the cardiac function was diagnosed by electrocardiogramanalysis, echocardiogram analysis and measurement of creatine kinase inblood.

By the echocardiogram analysis, the heart pump function was evaluatedbased on the ejection fraction (EF) of the heart as calculated by thefollowing formula:Ejection fraction (%)={(left ventricular relaxation terminaldiameter²−left ventricular contraction terminal diameter²)/leftventricular relaxation terminal diameter²)×100

The ejection fraction of healthy volunteers is known to be not less than60%.

The normal range of blood creatine kinase in healthy volunteers is knownto be 25 U/L-180 U/L for male, and 20 U/L-150 U/L for female.

Table 1 shows the results of the ejection fraction of the heart, themeasurement of blood creatine kinase and the above-mentioned three kindsof each marker. TABLE 1 Blood concentration Creatine Human H- MyosinLight Blood kinase FABP Chain I Troponin T sampling EF (%) (U/L) (ng/mL)(ng/mL) (ng/mL) First 53 37 6.8 <1.0 <0.01 time Second — 36 9.0 <1.00.01 time Third 37 33 19.6 <1.0 0.41 time—: no dataejection fraction of healthy volunteer: not less than 60%creatine kinase level of healthy volunteer: 25-180 U/L (male), 20-150U/L (female) cut-off value of acute myocardial infarction in thismeasurement;Human H-FABP: 6.2 ng/mL,Myosin Light Chain I: 2.5 ng/mL,Troponin T: 0.1 ng/mL

While not described in Table 1, in the electrocardiogram analysis ofthis patient, extension of QTc at about 2 weeks before the first bloodsampling was observed and T wave decreased at about 1 week before thethird blood sampling. In addition, sinus tachycardia andsupraventricular extrasystole were also observed then.

As shown in the above-mentioned Table 1, the ejection fraction was lowerthan the value of healthy volunteer (not less than 60%) in the firstblood sampling, and the toxicity to the heart due to adriamycin wasconsidered to have been expressed. However, the level of creatine kinasewas within the normal range, and the toxicity could not be detected. Atthis time point, of the three kinds of markers, only the level of humanH-FABP exceeded the above-mentioned cut-off value (6.2 ng/mL), and thelevels of Myosin Light Chain I and Troponin T were not more than themeasurement limits.

In the second blood sampling, the condition of the heart worsened ascompared to the first blood sampling and stronger toxicity is consideredto have expressed, in view of the findings from the electrocardiogramanalysis and the like. However, from the level of creatine kinase, thetoxicity could not be detected as in the previous time. At this timepoint, the human H-FABP level exceeded the above-mentioned cut-off valueand increased further. In contrast, the level of Myosin Light Chain Iwas not more than the measurement limit, and the level of Troponin T wasnot more than the cut-off value.

In the third blood sampling, the ejection fraction decreased to 37%, andthe condition of the heart was worse than in the first and the secondblood samplings, and still stronger toxicity is considered to haveexpressed. However, from the level of creatine kinase, the toxicitystill could not be detected. At this time point, the human H-FABP levelfar exceeded the above-mentioned cut-off value. In contrast, the levelof Myosin Light Chain I was not more than the measurement limit, and thelevel of Troponin T first exceeded the above-mentioned cut-off value.

EXAMPLE 2

Blood human H-FABP and the like of cancer patient who was administeredwith daunorubicin hydrochloride and diagnosed with cardiac failure dueto the expression of generalized edema were detected in the same manneras in the method described in Example 1. The results thereof are shownin the following Table 2. The second blood sampling in the Table wasperformed one month after the first blood sampling. TABLE 2 Bloodconcentration Creatine Human H- Myosin Light Blood kinase FABP Chain ITroponin T sampling (U/L) (ng/mL) (ng/mL) (ng/mL) First 11 7.6 <1.0<0.01 time Second 7 6.9 1.4 <0.01 timecreatine kinase level of healthy volunteer: 25-180 U/L (male), 20-150U/L (female)cut-off value of acute myocardial infarction in this measurement;Human H-FABP: 6.2 ng/mL,Myosin Light Chain I: 2.5 ng/mL,Troponin T: 0.1 ng/mL

The above-mentioned Table 2 reveals that, in the first and the secondblood samplings, human H-FABP alone exceeded the cut-off value (6.2ng/mL) of acute myocardial infarction, and other myocardial injurymarkers, i.e., Myosin Light Chain I and Troponin T, were within thenormal ranges. As shown in Table 2, creatine kinase did not exceed thelevel of healthy volunteer.

As shown in Table 1 and Table 2 above, of the three kinds of myocardialinjury markers, only human H-FABP sensitively detected expression of thecardiotoxicity due to adriamycin or daunorubicin hydrochloride.

Therefore, blood human H-FABP is a useful marker to predict the presenceand the level of cardiotoxicity caused by an anthracycline-typeanticancer chemotherapeutic agent such as adriamycin and the like.

INDUSTRIAL APPLICABILITY

According to the present invention, namely, a method of determiningcardiotoxicity caused by an anthracycline-type anticancerchemotherapeutic agent, which comprises detecting blood Human H-FABP,and a reagent therefor and the like, the cardiotoxicity can be detectedsensitively at an early stage, which enables physicians to conductmedical procedures at an early stage of cardiotoxicity expression, suchas change of pharmaceutical agents and the like.

This application is based on application No. 2002-093688 filed in Japan,the contents of which are incorporated hereinto by reference.

1. A method for determining toxicity to the heart of ananthracycline-type anticancer chemotherapeutic agent, which comprisesdetecting human H-FABP in the blood separated from human.
 2. The methodof claim 1, wherein the detection of human H-FABP is performed by animmunochemical method using an antibody that recognizes human H-FABP. 3.The method of claim 2, wherein the immunochemical method is an enzymeimmunochemical method, a latex agglutination assay or animmunochromatographic assay.
 4. The method of claim 2, wherein theantibody is a monoclonal antibody.
 5. The method of claim 1, wherein theanthracycline-type anticancer chemotherapeutic agent is adriamycin ordaunorubicin hydrochloride.
 6. A reagent for determining toxicity to theheart of an anthracycline-type anticancer chemotherapeutic agent, whichis used for performing the method of any of claims 1 to
 5. 7. A reagentfor determining toxicity to the heart of an anthracycline-typeanticancer chemotherapeutic agent, which comprises an antibody thatrecognizes human H-FABP.
 8. The reagent of claim 7, wherein the antibodyis a monoclonal antibody.
 9. The reagent of claim 7, wherein theanthracycline-type anticancer chemotherapeutic agent is adriamycin ordaunorubicin hydrochloride.
 10. A commercial package comprising thereagent of any of claims 7 to 9, and a written matter associatedtherewith, the written matter stating that said reagent can or should beused for determining toxicity to the heart of an anthracycline-typeanticancer chemotherapeutic agent.
 11. A kit for determining toxicity tothe heart of an anthracycline-type anticancer chemotherapeutic agent,which comprises an antibody that recognizes human H-FABP.
 12. The kit ofclaim 11, wherein the antibody is a monoclonal antibody.
 13. The kit ofclaim 11, wherein the anthracycline-type anticancer chemotherapeuticagent is adriamycin or daunorubicin hydrochloride.
 14. Use of anantibody that recognizes human H-FABP for determining toxicity to theheart of an anthracycline-type anticancer chemotherapeutic agent. 15.The use of claim 14, which comprises detecting human H-FABP in the bloodseparated from human.
 16. The use of claim 15, wherein the detection ofhuman H-FABP is performed by an enzyme immunochemical method, a latexagglutination assay or an immunochromatographic assay.
 17. The use ofclaim 14, wherein the antibody is a monoclonal antibody.
 18. The use ofclaim 14, wherein the anthracycline-type anticancer chemotherapeuticagent is adriamycin or daunorubicin hydrochloride.